This application is a continuation of our FIRST award with the same title. Our PROGRESS REPORT describes molecular genetic studies aimed at cloning and characterizing cDNAs for human and mouse methylmalonyl CoA mutase (MCM) cDNAs, the genomic loci (MUT, mut) encoding MCM, and mutations causing distinctive phenotypes of MCM deficiency. We have established methods for gene transfer into primary fibroblasts and S. ceriviseae and used these systems to characterize functional defects in mutant gene products and the biological response to overexpression of MCM. This proposal focuses on applying molecular methods to answer questions raised by our ongoing work concerning the dynamic role of MCM in metabolic flux, homeostasis, and disease. This work will focus on three questions. How do mutations in MCM affect enzyme activity? Mutation analysis has delineated two independent functional domains in MCM: one involved in cobalamin binding domain, and a second with undetermined function which participates in interallelic complementation. We will identify select mutations to assess the location and function of other domains within MCM and explore the relationship between the genotype and the biochemical or clinical phenotype of MCM deficiency. What mechanisms regulate the level of enzyme activity? Preliminary studies have identified several mechanisms which may determine the level of MCM activity. We will further assess the pattern of tissue specific expression of MCM mRNA as well as the role of sequences in the promoter and cDNA which may affect transcriptional or translational regulation of MCM. How does regulation of MCM enzyme activity and mutations in MCM affect metabolic flux through MCM? We have demonstrated that the level of MCM enzyme activity does not normally limit flux of metabolites from propionate through MCM in fibroblasts. We will study the relationship between MCM enzyme activity and metabolic flux through MCM in hepatoma cells by overexpressing recombinant MCM and inhibiting endogenous enzyme expression with antisense constructs. We will investigate whether MCM has a different kinetic role in metabolic flux from amino acids compared to propionate or methylmalonate. Finally, we will investigate the mechanism for the observation that some mutant alleles inhibit propionate flux through MCM in the presence of higher than normal levels of MCM enzyme activity; an observation which suggests that MCM may be a component of a larger multi-enzyme complex with a critical role in determining the capacity for propionate metabolism. These studies are intended to provide a dynamic understanding of the role of MCM in metabolic flux and homeostasis as well as the consequences of MCM deficiency.